The invention is generally in the field of immunotoxins, particularly immunotoxins effective against pathogenic cells, e.g., T lymphocytes mediating graft-versus-host-disease (GVHD).
Immunotoxins are molecules that contain targeting domains that direct the molecules to target cells of interest (e.g., effector T lymphocytes) and toxic domains that kill the target cells. They are thus useful in pathological conditions such as GVHD, cancer, autoimmune diseases, and certain infectious diseases. The field of immunotoxins has been limited by an inability to escalate the dose of immunotoxin administered to a subject to a level that is therapeutic but not unacceptably toxic.
The invention derives from the finding that treatment of animals undergoing lethal GVHD with a homodimeric immunotoxin abrogated the GVHD. Treatment with this immunotoxin allowed for a therapeutic window in that the dose of the dimeric immunotoxin required for abrogation of GVHD was more than four-fold lower than the toxic dose. The invention includes monomeric fusion proteins useful for the production of multimeric immunotoxic proteins, multimeric immunotoxic proteins, nucleic acid molecules encoding fusion protein monomers, vectors containing the nucleic acid molecules, and cells containing the vectors. Also included in the invention are in vitro and in vivo methods of killing a target cell involving delivery of a multimeric immunotoxic protein to the surface of a target cell and methods of producing both a fusion protein monomer of the invention and the multimeric immunotoxic proteins of the invention.
More specifically, the invention features a fusion protein molecule containing a toxic domain, a targeting domain, and at least one heterologous coupling moiety. In this fusion protein, cysteine residues forming disulfide bonds are cysteine residues native to the toxic domain or the targeting domain. Other aspects of the invention are: (a) an isolated nucleic acid molecule containing a nucleic acid sequence encoding the above fusion protein; (b) a vector containing the nucleic acid molecule of (a), e.g., a vector in which transcriptional regulatory elements (TRE) are operably linked to the nucleic acid sequence; (c) a cell containing the vector of (b); and (d) a method of making a fusion protein in which the cell of (c) is cultured and the fusion protein is extracted from the culture, i.e., either from the culture medium or from the cells. The invention also provides a multimeric immunotoxic protein containing at least two of the above fusion protein molecules, each fusion protein molecule being joined by at least one of the heterologous coupling moieties to one or more of the other fusion protein molecules.
In addition, the invention encompasses a multimeric immunotoxic protein containing at least two fusion protein monomers, each of which includes a targeting domain and a toxic domain and is physically associated with the other fusion protein monomers. The targeting domain in all the multimeric immunotoxic proteins of the invention have significant binding affinity for a target molecule on a target cell. The fusion protein monomers can contain one or more coupling moieties and the physical association of the fusion protein monomer to one or more other fusion protein monomers can be mediated by at least one of the coupling moieties. The coupling moiety can be a terminal moiety, i.e., a C-terminal moiety or a N-terminal moiety. A coupling moiety can be, for example, a cysteine residue. Furthermore the coupling moieties can be heterologous coupling moieties. The fusion protein monomers in a particular multimeric immunotoxic protein can have the same amino acid sequence or different amino acid sequences. Targeting domains can be antibody fragments, e.g., single chain Fv and can have significant binding affinity for a target molecules on a T cell, e.g., a CD3 polypeptide. Alternatively, the targeting domain can be, for example, a polypeptide such as a cytokine, a ligand for a cell adhesion receptor, a ligand for a signal transduction receptor, a hormone, a molecule that binds to a death domain family molecule (e.g., Fas ligand, TNF-alpha, or TWEAK), an antigen, or a functional fragment of any of these polypeptides. The toxic domain can be, for example, any of the following toxic polypeptides: ricin, Pseudomonas exotoxin (PE), bryodin, gelonin, xcex1-sarcin, aspergillin, restrictocin, angiogenin, saporin, abrin, pokeweed antiviral protein (PAP), or a functional fragment of any of these toxic polypeptides. The toxic domain can also be diphtheria toxin (DT) or a functional fragment thereof, e.g., a fragment containing amino acid residues 1-389 of DT. The target cell to which the multimeric immunotoxic proteins of the invention bind can be in a mammal. The mammal can be one suspected of having graft-versus-host disease (GVHD). A target cell to which the multimeric immunotoxic proteins bind can be a cancer cell, e.g., a neural tissue cancer cell, a melanoma cell, a breast cancer cell, a lung cancer cell, a gastrointestinal cancer cell, an ovarian cancer cell, a testicular cancer cell, a lung cancer cell, a prostate cancer cell, a cervical cancer cell, a bladder cancer cell, a vaginal cancer cell, a liver cancer cell, a renal cancer cell, a bone cancer cell, and a vascular tissue cancer cell.
The invention also features a method of killing a target cell in which the target cell is contacted with any of the multimeric proteins of the invention. The contacting can be in vitro or the target cell can be in a mammal. Where the target cell is in a mammal, the multimeric immunotoxic protein per se can administered to the mammal. Alternatively, one or more nucleic acids encoding at least two fusion protein monomers can be administered to the mammal. In addition, the multimeric immunotoxic proteins can be delivered to a target cell by an ex vivo methodology which can, for example, involve: (a) providing a recombinant cell which is the progeny of a cell from the mammal or from another mammal and has been transfected or transformed ex vivo with a vector containing one or more nucleic acid sequences, each nucleic acid sequence encoding a fusion protein monomer with a different amino acid sequence, such that the recombinant cell expresses the multimeric protein; and (b) administering the recombinant cell to the mammal.
Also within the invention is a method of making a multimeric immunotoxic protein. The method can involve the steps of: (a) providing one or more cells, each of the cells containing a nucleic acid sequence encoding a fusion protein monomer composed of a targeting domain and a toxic domain, each monomer having a different amino acid sequence, and the nucleic acid sequence being operably linked to a TRE; (b) separately culturing each of the one or more cells; (c) extracting the fusion protein monomer from each of the cultures (cells or medium); (d) mixing the fusion protein monomers; and (e) exposing the mixed fusion protein monomers to conditions which allow multimerization of the fusion protein monomers.
xe2x80x9cPolypeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.
As used herein, xe2x80x9coperably linkedxe2x80x9d means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
As used herein, the term xe2x80x9cantibody fragmentsxe2x80x9d refers to antigen-binding fragments, e.g., Fab, F(abxe2x80x2)2, Fv, and single chain Fv fragments. Also included are chimeric antibody fragments in which the regions involved in antigen binding (e.g., complementarity determining regions (CDR) 1, 2, and 3) are from an antibody produced in a first species (e.g., a mouse or a hamster) and the regions not involved in antigen binding (e.g., framework regions) are from an antibody produced in a second species (e.g., a human).
As used herein, a xe2x80x9cfunctional fragmentxe2x80x9d of a toxic polypeptide for use as a toxic domain in the fusion proteins of the invention is a fragment of the toxic polypeptide shorter than the full-length, wild-type toxic polypeptide but which has at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even more) of the toxic activity of the full-length, wild-type toxic polypeptide. In vitro and in vivo methods for comparing the relative toxic activity of two or more test compounds are known in the art.
As used herein, a xe2x80x9cfunctional fragmentxe2x80x9d of a targeting polypeptide for use as a targeting domain in the fusion proteins of the invention is a fragment of the targeting polypeptide shorter than the full-length,wild-type targeting polypeptide but which has at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even more) of the ability of the full-length, wild-type targeting polypeptide to bind to its relevant target molecule. Methods of comparing the relative ability of two or more test compounds to bind to a target molecule are well-known to artisans in the field, e.g., direct or competitive ELISA.
As used herein, a xe2x80x9ccoupling moietyxe2x80x9d in a fusion protein of the invention is a molecule that can be, but is not necessarily, an amino acid (e.g., cysteine or lysine), and which is inserted either internally or at a terminus (C or N) of the fusion protein. Coupling moieties can be residues that are present in native polypeptides (or functional fragments thereof) used as targeting or toxic domains or they can be heterologous. Coupling moieties serve as sites for joining of one fusion protein to another.
As used herein, a xe2x80x9cheterologous moietyxe2x80x9d in a polypeptide is a moiety that does not occur in the wild-type form(s) of the polypeptide or functional fragment(s) thereof.
As used herein, xe2x80x9cphysically associatedxe2x80x9d fusion proteins are fusion proteins that are either: (a) directly joined to each other by, for example, a covalent bond or interactions such as hydrophobic interactions or ionic interactions; or (b) are indirectly linked to each other by one or more intervening fusion proteins, each linked in a sequential fashion by the above bond or interactions.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
Other features and advantages of the invention, e.g., abrogating GVHD in mammalian subjects, will be apparent from the following description, from the drawings and from the claims.